Pancreas
The pancreas is an elongated, tapered organ which lies to the rear of the upper left hand side of the abdominal cavity. It has been anatomically described as containing three main sections including a head (widest end—located near the duodenum), a body, and a tail (tapered end—located near the spleen). This organ houses two main tissue types: exocrine tissue, comprised of both acinar and ductal cells; and endocrine tissue, containing cells which produce hormones (i.e., insulin) for delivery into the bloodstream. The exocrine pancreas, comprising about 95% of the pancreatic mass, is an acinar gland containing clusters of pyramidal secretory cells (referred to as acini) that produce digestive enzymes (i.e., amylase, lipase, phospholipase, trypsin, chymotrypsin, aminopeptidase, elastase and various other proteins). These enzymes are delivered to the digestive system by tubes constructed of cuboidal ductal cells, which also produce bicarbonate for digestive purposes. Between the secretory acini and ductal tubes is located a connecting cell component referred to as centroacinar cells.
The endocrine pancreas, comprising only about 1-2% of the pancreatic mass, contains clusters of hormone-producing cells referred to as islets of Langerhans (the islet cells are responsible for the maintenance of blood glucose levels by secreting insulin). These clusters are made up of at least seven cell types, including, but not limited to, insulin-producing β-cells, glucagon-producing α-cells, somatostatin-producing δ-cells, and PP-cells which produce pancreatic polypeptide (Edlund, H., 2002). In addition, a subpopulation of endocrine cells referred to as ε-cells recently has been described (Heller, R. S., et al., 2005). These cells were discovered based on their production of ghrelin, an appetite stimulating peptide known to be secreted by enteroendocrine cells of the digestive tract.
Transcriptional Cascade Underlying Endocrine Pancreas and β-Cell Differentiation
Endoderm specification, foregut and midgut endoderm specification and subsequently pancreas specification are regulated by a complement of transcription factors (FIG. 1). Specifically, initial endoderm specification in the mouse involves expression of Sox17 (Kanai-Azuma, M. et al., 2002), as well as Gata-5 and Gata-6 (Weber, H. et al., 2000; Bossard, P., and Zaret, K. S. 1998) and Mixer/Mix.3 (Henry, G. L., and Melton, D. A. 1998). Subsequently, the hepatocyte nuclear factor, Hnf3β/Foxa2, is needed for the development of prospective foregut and midgut endoderm (Ang, S. L., et al., 1993). Other transcription factors then commit the foregut and midgut endoderm to liver, thyroid, lung, gastric, duodenal and pancreas endoderm.
In the mouse, pancreas is derived in part from the ventral and dorsal foregut endoderm, which subsequently fuse to form the mature organ. Commitment to the pancreas is associated with expression of the transcription factors Hlxb9 and Pdx-1. Deletion of Hlxb9 (Hentsch, B. et al., 1996) or Pdx-1 (Offield, M. F. et al., 1996) leads to dorsal or complete pancreas agenesis, respectively, even though a dorsal pancreas bud can be detected in Pdx-1 deficient embryos. Ventral pancreas formation is relatively normal in Hlxb9 deficient embryos, whereas dorsal pancreas specification is deficient.
These phenotypes suggest that initial specification is different between dorsal and ventral pancreas. As a pancreatic bud is still formed, despite the elimination of either transcription factor, other signals may be present before expression of Hlxb9 or Pdx-1 for pancreatic commitment. Further commitment to exocrine versus endocrine pancreas is associated with expression of Ptf1a/p48 (Ahlgren, U. et al., 1998) and Ngn3 (Gradwohl, G. et al., 2000), respectively. Of note, Ptf1a/p48 appears to also be needed earlier, i.e., during specification of the ventral pancreatic bud (Kawaguchi, Y. et al., 2002). Like Pdx-1, which is needed to specify pancreatic endoderm, Ngn3 is needed to specify pancreatic endoderm to the endocrine lineage, and it is believed that endocrine cells are derived from Ngn3 expressing cells. Ngn3 is also expressed in the central nervous systems (CNS), and deletion of this transcription factor not only affects endocrine pancreas development, but also nervous system development. Further commitment to β-cells in vivo is associated with expression of Pax4 (Sosa-Pineda, B. et al., 1997), Pax6 (Sander, M. et al., 1997), Nkx2.2 (Sussel, L. et al., 1998; accession number NM—002509 for human mRNA sequence) and Nkx6.1 (Sander, M. et al., 2000).
Extracellular Signals Underlying Endocrine Pancreas and β-Cell Differentiation
During development endoderm is specified by a combination of factors, including members of the TGFβ and Wnt family. Wnt3 is expressed in the primitive streak and developing mesoderm, and Wnt3 null mice do not form mesoderm or endoderm (Liu, P. et al., 1999). Nodal is expressed in the epiblast and in the anterior regions of the primitive streak (Zhou, X. et al., 1993), and like Wnt3 null embryos, Nodal null embryos also fail to develop mesoderm and endoderm. Using Xenopus animal cap assays, it was also shown that activin-A, another member of the TGF family, induces both mesoderm and endoderm specification in a dose dependent fashion, with high concentrations of activin-A inducing dorsal mesoderm and endoderm and low concentrations inducing ventral mesoderm (McDowell, N. et al., 1997).
Subsequent pancreas commitment and endocrine pancreas commitment is also regulated by members of the TGFβ and Wnt family, as well as by members of the FGF and hedgehog families. Compared with initial endoderm specification, which requires among other signals Wnt3, Wnts may inhibit pancreatic endoderm specification. Indeed, expression of Wnt1 or Wnt5a under the control of the Pdx-1 promoter alters the foregut region, which now resembles a posterior extension of the stomach rather than normally comprising the proximal duodenum, and is associated with reduction or complete agenesis of the pancreas. Consistent with this observation, several Wnt signaling inhibitors can be detected in the mouse embryonic pancreas, including sFRP-1, -2, -3 and -4 as well as Dkks (Heller, R. S. et al., 2002). Pancreas commitment from the ventral as well as dorsal forgut endoderm is inhibited by sonic hedgehog (SHH) (Hebrok, M. et al., 2000). Elimination of the SHH receptor, patched (Ptc), causes more widespread differentiation to pancreatic epithelium. It is thought that activin-A (Maldonado, T. S. et al., 2000) and/or FGF2 (Hardikar, A. A. et al., 2003) signals from the notochord act to repress SHH expression in pre-pancreatic endoderm.
Pancreas versus liver specification in the ventral gut endoderm is at least in part determined by FGF2 produced by the adjacent cardiac mesoderm (Jung, J. et al., 1999), which suppresses pancreas specification, whereas low doses of FGF2 may be important for pancreas differentiation from dorsal foregut endoderm (Hardikar, A. A. et al., 2003). In addition, pancreas specification and differentiation is regulated by Notch signaling (Jensen, J. et al., 2000). Elimination of Notch pathway components, such as Dll-1 or Hes-1, leads to accelerated differentiation to pancreas epithelium.
Endocrine versus exocrine pancreas differentiation is regulated by endoderm-mesoderm interactions (Gittes, G. K. et al., 1996), in part mediated by cell-extracellular matrix (ECM) interactions and by members of the BMP family of growth factors, including activin and TGFβ. Endodermal-mesenchymal interactions have a dual role in endocrine pancreas differentiation. These interactions are key between E9.5 and 10.5 for inducing pancreas commitment, whereas interactions between pancreas committed endoderm and laminin, produced by the mesenchyme subsequently steers differentiation into an exocrine phenotype (Sanvito, F. et al., 1994). In addition, TGFβ members, such as BMP2, produced by the mesenchyme, may prevent endocrine specification while favoring exocrine pancreas differentiation in vivo. FGFs produced by mesenchymal cells, such as FGF10, also play a role. FGF10 appears to play a role in proliferation of Pdx-1+ pancreatic progenitors (Bhushan, A. et al., 2001).
Diabetes
Diabetes mellitus is a medical condition characterized by variable yet persistent high blood-glucose levels (hyperglycemia). Diabetes is a serious devastating illness that is reaching epidemic proportions in both industrialized and developing countries. In 1985, there were approximately 30 million people with diabetes worldwide, which increased 135 million in 1995 and is expected to increase further by close to 50% by 2050. Diabetes is the fifth leading cause of death in the United States. According to the American Diabetes Association, the economic cost of diabetes in the U.S. in 2002 was $132 billion, including $92 billion of direct costs. This figure is expected to reach in excess of $190 billion by 2020.
Generally, diabetes mellitus can be subdivided into two distinct types: Type 1 diabetes and Type 2 diabetes. Type 1 diabetes is characterized by little or no circulating insulin and it most commonly appears in childhood or early adolescence. It is caused by the destruction of the insulin-producing beta cells of the pancreatic islets. To survive, people with Type 1 diabetes must take multiple insulin injections daily and test their blood sugar multiple times per day. However, the multiple daily injections of insulin do not adequately mimic the body's minute-to-minute production of insulin and precise control of glucose metabolism. Blood sugar levels are usually higher than normal, causing complications that include blindness, renal failure, non-healing peripheral vascular ulcers, the premature development of heart disease or stroke, gangrene and amputation, nerve damage, impotence and it decreases the sufferer's overall life expectancy by one to two decades.
Type 2 diabetes usually appears in middle age or later and particularly affects those who are overweight. In Type 2 diabetes, the body's cells that normally require insulin lose their sensitivity and fail to respond to insulin normally. This insulin resistance may be overcome for many years by extra insulin production by the pancreatic beta cells. Eventually, however, the beta cells are gradually exhausted because they have to produce large amounts of excess insulin due to the elevated blood glucose levels. Ultimately, the overworked beta cells die and insulin secretion fails, bringing with it a concomitant rise in blood glucose to sufficient levels that it can only be controlled by exogenous insulin injections. High blood pressure and abnormal cholesterol levels usually accompany Type 2 diabetes. These conditions, together with high blood sugar, increase the risk of heart attack, stroke, and circulatory blockages in the legs leading to amputation.
There is a third type of diabetes in which diabetes is caused by a genetic defect, such as Maturity Onset Diabetes of the Young (MODY). MODY is due to a genetic error in the insulin-producing cells that restricts its ability to process the glucose that enters this cell via a special glucose receptor. Beta cells in patients with MODY cannot produce insulin correctly in response to glucose, resulting in hyperglycemia and require treatment that eventually also requires insulin injections.
The currently available medical treatments for insulin-dependent diabetes are limited to insulin administration, pancreas transplantation (either with whole pancreas or pancreas segments) and pancreatic islet transplantation. Insulin therapy is by far more prevalent than pancreas transplantation and pancreatic islet transplantation. However, controlling blood sugar is not simple. Despite rigorous attention to maintaining a healthy diet, exercise regimen, and always injecting the proper amount of insulin, many other factors can adversely affect a person's blood-sugar control including: stress, hormonal changes, periods of growth, illness or infection and fatigue. People with diabetes must constantly be prepared for life threatening hypoglycemic (low blood sugar) and hyperglycemic (high blood sugar) reactions.
In contrast to insulin administration, whole pancreas transplantation or transplantation of segments of the pancreas is known to have cured diabetes in patients. However, due to the requirement for life-long immunosuppressive therapy, the transplantation is usually performed only when kidney transplantation is required, making pancreas-only transplantations relatively infrequent operations. Although pancreas transplants are very successful in helping people with insulin-dependent diabetes improve their blood sugar to the point they no longer need insulin injections and reduce long-term complications, there are a number of drawbacks to whole pancreas transplants. Most importantly, getting a pancreas transplant involves a major operation and requires the use of life-long immunosuppressant drugs to prevent the body's immune system from destroying the pancreas that is a foreign graft. Without these drugs, the pancreas is destroyed in a matter of days. The risks in taking these immunosuppressive drugs is the increased incidence of infections and tumors that can both be life threatening.
Pancreatic islet transplants are much simpler and safer procedures than whole pancreas transplants and can achieve the same effect by replacing beta cells. However, the shortage of islet cells available for transplantation remains an unsolved problem in islet cell transplantation. Since islets form only about 2% of the entire pancreas, isolating them from the rest of the pancreas that does not produce insulin takes approximately 6 hours. Although an automated isolation method has made it possible to isolate enough islets from one pancreas to transplant into one patient, as opposed to the 5 or 6 organs previously needed to carry out one transplant, the demand for islets still exceeds the currently available supply of organs harvested from cadavers. Additionally, long term resolution of diabetic symptoms is often not achieved.
An alternative to insulin injections, pancreas transplantation and pancreatic islet transplantation would fulfill a great public health need.
Stem Cells
The embryonic stem (ES) cell has unlimited self-renewal and can differentiate into all tissue types. ES cells are derived from the inner cell mass of the blastocyst or primordial germ cells from a post-implantation embryo (embryonic germ cells or EG cells). ES (and EG) cells can be identified by positive staining with antibodies to SSEA 1 (mouse) and SSEA 4 (human). At the molecular level, ES and EG cells express a number of transcription factors specific for these undifferentiated cells. These include Oct-4 and rex-1. Rex expression depends on Oct-4. Also found are LIF-R (in mouse) and the transcription factors sox-2 and rox-1. Rox-1 and sox-2 are also expressed in non-ES cells. Another hallmark of ES cells is the presence of telomerase, which provides these cells with an unlimited self-renewal potential in vitro.
The ability to generate functional islet cells from a long-term expandable stem cell population would provide a source of β-cells for transplantation in patients with diabetes. One such population under consideration is embryonic stem (ES) cells. When embryonic stem cells are allowed to form embryoid bodies in vitro, rare cells with β-cell characteristics can be detected amongst the endodermal cell types. Recent studies have demonstrated that relative specific differentiation of mouse and human ES cells to hepatic or pancreatic endoderm may be possible. Treatment with high concentrations of activin has resulted in the specification of ES cells to endoderm (Kubo, A. et al., 2004). A number of studies have also suggested that insulin-positive cells can be obtained from ES cells using a number of different strategies (Lumelsky, N. et al., 2001; Hori, Y. et al., 2002; Soria, B. et al., 2000; Kahan, B. W. et al., 2003). However, some of these studies did not address whether insulin that was detected was insulin-1 or insulin-2, the latter also found in neural cells and extra-embryonic endoderm (Sipione S., et al., 2004). An additional complication is that most studies cultured ES cells in insulin containing medium, and several groups have now shown that insulin may be absorbed by cells from the medium (Vaca P. et al., 2005; Rajagopal J. et al., 2003; Hansson M. et al., 2004). An additional problem, to be overcome for ES cell-derived β-like cells to be used in the clinic, is the ability of undifferentiated ES cells, even when present in low numbers, to cause teratoma formation (Bjorklund et al., 2002).
As diabetes reaches an epidemic status worldwide, a need for novel and curative therapies is evident. With the advent of islet transplantation as a potential therapy for type-1 diabetes, the paucity of donor pancreata has become a limiting factor. Thus, there is a need for an abundant, clinically relevant, cell source for use as an alternative to insulin injections, pancreas transplantation and pancreatic islet transplantation.
“Multipotent adult progenitor cells” (MAPCs) are non-embryonic (non-ES), non-germ and non-embryonic germ (non-EG) cells that can differentiate into one or more ectodermal, endodermal and mesodermal cells types. MAPCs can be positive for telomerase, Oct-3A (Oct-3/4) or a combination thereof. Telomerase or Oct-3/4 have been recognized as genes that are primary products for the undifferentiated state. Telomerase is needed for self renewal without replicative senescence. MAPCs derived from human, mouse, rat or other mammals appear to be the only normal, non-malignant, somatic cell (i.e., non-germ cell) known to date to express telomerase activity even in late passage cells. The telomeres are not sequentially reduced in length in MAPCs. MAPCs are karyotypically normal. MAPCs may also express SSEA-4 and nanog.
The Oct-4 gene (Oct-3 in humans) is transcribed into at least two splice variants in humans, Oct-3A and Oct-3B. The Oct-3B splice variant is found in many differentiated cells, whereas the Oct-3A splice variant (also previously designated Oct 3/4) is reported to be specific for the undifferentiated embryonic stem cell (Shimozaki et al. 2003). Oct-4 (Oct-3 in humans) is a transcription factor expressed in the pregastrulation embryo, early cleavage stage embryo, cells of the inner cell mass of the blastocyst, and embryonic carcinoma (EC) cells (Nichols J., et al 1998), and is down-regulated when cells are induced to differentiate. Expression of Oct-4 plays an important role in determining early steps in embryogenesis and differentiation. Oct-4, in combination with Rox-1, causes transcriptional activation of the Zn-finger protein Rex-1, also required for maintaining ES in an undifferentiated state (Rosfjord and Rizzino A. 1997; Ben-Shushan E, et al. 1998). In addition, sox-2, expressed in ES/EC, but also in other more differentiated cells, is needed together with Oct-4 to retain the undifferentiated state of ES/EC (Uwanogho D et al. 1995). Maintenance of murine ES cells and primordial germ cells requires LIF.
MAPCs have the ability to regenerate all primitive germ layers (endodermal, mesodermal and ectodermal) in vitro and in vivo. In this context they are equivalent to embryonic stem cells and distinct from mesenchymal stem cells, which are also isolated from bone marrow. The biological potency of MAPCs has been proven in various animal models, including mouse, rat, and xenogeneic engraftment of human stem cells in rats or NOD/SCID mice (Reyes, M. and C. M. Verfaillie 2001; Jiang, Y. et al. 2002). Clonal potency of this cell population has been shown. Single genetically marked MAPCs were injected into mouse blastocysts, blastocysts implanted, and embryos developed to term (Jiang, Y. et al. 2002). Post-natal analysis in chimeric animals showed reconstitution of all tissues and organs, including liver. Dual staining experiments demonstrated that gene-marked MAPCs contributed to a significant percentage of apparently functional cardiomyocytes in these animals. These animals did not show any heart abnormalities or irregularities in either the embryological or adult state. No abnormalities or organ dysfunction were observed in any of these animals.
MAPCs are capable of extensive culture without loss of differentiation potential and show efficient, long term, engraftment and differentiation along multiple developmental lineages in NOD-SCID mice, without evidence of teratoma formation (Reyes, M. and C. M. Verfaillie 2001). This includes endothelial lineage differentiation (Verfaillie, 2002; Jahagirdar, et al. 2001).